ESTROGEN RECEPTOR MEDIATED REPROGRAMMING OF THE PROSTATE IN BPH Paul S. Cooke and Gail S Prins, Multi-PIs Abstract Exposure to estrogens during critical developmental periods can permanently reprogram the prostate gland, resulting in growth abnormalities in adult life that include stromal and epithelial hyperplasia, benign adenomas and chronic prostatitis. As such, we propose that early-life estrogenic exposures may be a predisposing factor for benign prostatic hyperplasia (BPH) in aging males. Past work has established that epigenetic modifications underpin developmental reprogramming of the prostate; however, the pathways that lead to this epigenomic reorganization are unclear. We previously showed that estrogen receptor 1 (ESR1; also known as ER?) is essential and sufficient for this reprogramming; however, distinct signaling pathways initiated through membrane ESR1 (mESR1) or nuclear ESR1 (nESR1) actions have not been clarified. Our new published and preliminary data now reveal essential roles for mESR1 in normal male reproductive development and fertility, as well as the normal prostatic response to developmental estrogenization. Further, a recent report found that important epigenetic changes that could lead to hyperresponsivity of certain genes involved in growth and cell proliferation can be initiated through mESR1 signaling cascades in the developing prostate gland. In this context, the goals of the proposed research are to delineate relative roles of mESR1 and nESR1 in mediating developmental estrogen reprogramming and to identify ?nongenomic? pathways utilized by mESR1 to reprogram epigenomic memory within the gland, including prostate epithelial stem cells. Herein, we will take an innovative approach to directly interrogate mESR1 and nESR1 actions by utilizing newly developed knock-in mouse models that express nESR1 normally but lack mESR1 signaling (nuclear-only ESR1 mouse; NOER) or that express mESR1 and its downstream signaling pathways but lack nESR1 (H2NES mouse). These powerful and unique new models allow us to take a direct experimental approach to tease out the specific role(s) for ESR1 in each compartment and their downstream effectors on epigenetic imprinting in the prostate, which has previously not been possible. Three Specific Aims are proposed to accomplish these goals. Aim 1: Establish whether mESR1 is necessary and/or sufficient for estrogen-driven developmental prostate reprogramming. Aim 2: Identify signaling cascades involved in estrogen-induced epigenetic imprinting. Aim 3: Elucidate the roles of mESR1 and nESR1 in reprogramming prostate stem/progenitor cells. The proposed studies will fill knowledge gaps on the differential roles of mESR1 and nESR1 in prostate development and broaden the mechanistic basis for estrogenic imprinting in the prostate. Together, the results will provide new and clinically relevant insights regarding the role of estrogen signaling through both mESR1 and nESR1 in driving BPH and inform future drug discovery strategies that can target membrane-initiated estrogen signaling in the prostate.